胡黄连苷Ⅱ干预脑缺血/再灌注损伤后p38 丝裂原活化蛋白激酶信号通路的作用机制

doi:10.16098/j.issn.0529-1356.2021.02.006

摘要/Abstract

摘要：

目的探讨胡黄连苷Ⅱ对脑缺血/再灌注损伤后p38丝裂原活化蛋白激酶(p38 MAPK)通路的影响及其神经保护作用机制。方法成年健康雄性Wistar大鼠150只，应用线栓法建立大鼠大脑中动脉闭塞/再灌注（MCAO/R）模型，按照随机对照原则，动物分为假手术组、模型组、胡黄连苷组、茴香霉素（p38 MAPK激活剂）组、茴香霉素+胡黄连苷组和SB203580（p38 MAPK抑制剂）组和SB203580+胡黄连苷组。改良神经功能评分（mNSS）法评价大鼠神经行为功能，HE染色观察神经细胞的形态结构， TUNEL法检测细胞凋亡，免疫组织化学法检测脑组织磷酸化p38 MAPK(p-p38 MAPK)表达水平，Western blotting检测大鼠皮质区p-p38 MAPK、磷酸化MAPK激活的蛋白激酶2（p-MK2）、磷酸化胞质磷脂酶A2（p-cPLA2）、白细胞介素6（IL-6）和肿瘤坏死因子α（TNF-α）。结果假手术组大鼠无神经功能缺损。模型组大鼠神经功能缺损评分升高，皮质区神经细胞损伤加重，脑梗死体积增大，凋亡细胞增多，p-p38 MAPK、p-MK2、p-cPLA2、IL-6及TNF-α蛋白表达增强。胡黄连苷组、SB203580组和SB203580+胡黄连苷组大鼠皮质区神经元损伤较轻，凋亡细胞数量明显减少，p-p38 MAPK、p-MK2、p-cPLA2以及IL-6蛋白表达较模型组明显降低。茴香霉素组、茴香霉素+胡黄连苷组各项指标与模型组相近，脑梗死体积较大，神经功能缺损较重，p-p38 MAPK、p-MK2、p-cPLA2以及IL-6蛋白表达升高。结论脑缺血损伤后激活p38 MAPK信号通路介导神经元凋亡和炎症反应，胡黄连苷Ⅱ可能通过降低p38 MAPK通路的活化，抑制神经元凋亡和炎症反应从而保护神经系统。

关键词: 胡黄连苷Ⅱ, 脑, 缺血/再灌注损伤, p38丝裂素活化蛋白激酶信号通路, 免疫组织化学, 免疫印迹法, 大鼠

Abstract:

Objective To explore the neuroprotective effect and mechanism of picroside Ⅱ on p38 mitogen activated protein kinase（p38 MAPK）signal transduction pathway after cerebral ischemia/reperfusion injury in rats. Methods A total of 150 healthy male Wistar rats were subject to establish middle cerebral artery occlusion/reperfusion (MCAO/R) models by inserting a monofilament thread. All rats were randomly divided into sham group, model group, picroside (Picr) group, anisomycin (Anis，agonist of p38 MAPK) group, Anis+Picr group, SB203580 (SB, inhibitor of p38 MAPK) group and SB+Picr group. The neurobehavioral function was evaluated by modified neurological severity score points (mNSS) test. The structure of neuron was observed using HE staining. The apoptotic cells were counted using TUNEL assay. The expression of phosphorylated p38 MAPK (p-p38 MAPK) in cortex was determined using the immunohistochemistry. And the expressions of p-p38 MAPK, phosphorylated MAPK activated protein kinase-2 (p-MK2), phosphorylated cytoplasm phospholipase A2 (p-cPLA2), interleukin-6 (IL-6) and tumor necrotic factor α (TNF-α) were determined by Western blotting. Results No neurological behavioral malfunction was found in sham group. In model group, the damage of neuron was worsened, while the neurobehavioral function score, apoptotic cell index and the expressions of p-p38 MAPK, p-MK2, p-cPLA2，IL-6 and TNF-α increased significantly than those in control group. No significant difference was found in TNF-α. In Picr group, SB group and SB+Picr group, the damage of neuron was lighter, the neurological behavioral function was improved, the number of apoptotic cells and the expressions of p-p38 MAPK, p-MK2, p-cPLA2 and IL-6 decreased significantly than those in model group. In Anis group and Anis+Picr group, the damage was worsen, the cerebral infarction was larger, and the expressions of p-p38 MAPK, p-MK2, p-cPLA2 and IL-6 increased significantly than those in control group. Conclusion Picroside Ⅱ can protect the neuron from the apoptosis and inflammation reaction after MCAO/R by inhibiting p38 MAPK signal transduction pathway in rats.

Key words:

Picroside Ⅱ, Brain, Ischemia/reperfusion injury, p38 mitogen activated protein kinase signal transduction pathway, Immunohistochemistry, Western blotting, Rat

中图分类号:

Q421

于竹芹汪贯习王潇璐王悦王婷婷. 胡黄连苷Ⅱ干预脑缺血/再灌注损伤后p38 丝裂原活化蛋白激酶信号通路的作用机制[J]. 解剖学报, 2021, 52(2): 196-204.

YU Zhu-qin WANG Guan-xi WANG Xiao-lu WANG Yue WANG Ting-ting. Effect and mechanism of picroside Ⅱ on p38 mitogen activated protein kinase signal transduction pathway after cerebral ischemia/reperfusion in rats[J]. Acta Anatomica Sinica, 2021, 52(2): 196-204.

参考文献

［1］ Liu L, Wang D, Wong KS, et al. Stroke and stroke care in China: huge burden, significant workload, and a national priority［J］. Stroke, 2011, 42(12):3651-3654.

［2］ Wang P, Miao CY. NAMPT as a therapeutic target against stroke［J］. Trends Pharmacol Sci, 2015, 36(12): 891-905.

［3］ Sui X, Kong N, Ye L, et al. p38 and JNK MAPK pathways control the balance of apoptosis and autophagy in response to chemotherapeutic agents［J］. Cancer Lett, 2014, 344(2): 174-179.

［4］ Raingeaud J, Gupta S, Rogers JS, et al. Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine［J］. J Biol Chem, 1995, 270(13):7420-7426.

［5］ Cuadrado A, Nebreda AR. Mechanisms and functions of p38 MAPK signalling［J］. Biochem J, 2010, 429(3): 403-417.

［6］ Wu DC, Ye W, Che XM, et al. Activation of mitogen activated protein kinases after permanent cerebral artery occlusion in mouse brain［J］. J Cereb Blood Flow Metab, 2000, 20(9): 1320-1330．

［7］ Porrini V, Sarnico I, Benarese M, et al. Neuroprotective and anti-apoptotic effects of CSP-1103 in primary cortical neurons exposed to oxygen and glucose deprivation［J］. Int J Mol Sci, 2017, 18(1):184.

［8］ Lockhart C. Neuroprotective effect of muscone on glutamate-induced apoptosis in PC12 cells via antioxidant and Ca(2+) antagonism［J］. Neurochem Int, 2014, 70(1):10-21.

［9］ Wang CP, Shi YW, Tang M, et al. Isoquercetin ameliorates cerebral impairment in focal ischemia through anti-oxidative, anti-inflammatory, and antiapoptotic effects in primary gulture of rat hippocampal neurons and hippocampal CA1 region of rats［J］. Mol Neurobiol, 2017, 54(3):2126-2142.

［10］ Zhang T, Fang S, Wan C, et al. Excess salt exacerbates blood-brain barrier disruption via a p38/MAPK/SGK1-dependent pathway in permanent cerebral ischemia［J］. Sci Rep, 2015, 5:16548.

［11］ Xue LX, Xu ZH, Wang JQ, et al. Activin A/Smads signaling pathway negatively regulates oxygen glucoserivation-induced autophagy via suppression of JNK and p38 MAPK pathways in neuronal PC12 cells［J］. Biochem Biophys Res Commun, 2016, 480(3):355-361.

［12］ Zhan L, Liu L, Li K, et al. Neuroprotection of hypoxic postconditioning against global cerebral ischemia through influencing posttranslational regulations of heat shock protein 27 in adult rats［J］. Brain Pathol, 2017, 27(6): 822-838.

［13］ Guo YL, Xu XL, Li Q, et al. Anti-inflammation effects of picroside 2 in cerebral ischemic injury rats［J］. Behav Brain Funct, 2010, 6(1):43-49.

［14］ Li Zh, Li Q, Guo YL, et al.Interference effect of picroside Ⅱ on cerebral ischemia reperfusion injury in rats［J］.Acta Anatomica Sinica,2010,41(1)：9-12.（in Chinese）

李震, 李琴, 郭云良, 等. 胡黄连苷Ⅱ对大鼠脑缺血再灌注损伤的干预作用［J］. 解剖学报, 2010, 41(1):9-12.

［15］ Sugino T, Nozaki K, Takagi Y, et al. Activation ofmitogen-activatedprotein kinases after transient forebrain ischemia in gerbilhippocampus［J］. J Neurosci, 2000, 20 (12):4506-4514.

［16］ Wang W, Tang L, Li Y, et al. Biochanin A protects against focal cerebral ischemia/reperfusion in rats via inhibition of p38-mediated inflammatory responses［J］. J Neurol Sci, 2014, 348(1-2): 121-125.

［17］ Nito C, Kamada H, Endo H, et al. Role of the p38 mitogen-activated protein kinase/cytosolic phospholipase A2 signaling pathway in bloodbrain barrier disruption after focal cerebral ischemia and reperfusion［J］. J Cereb Blood Flow Metab, 2008, 28(10):1686-1696.

［18］ Kishimoto K, Li RC, Zhang J, et al. Cytosolic phospholipase A2 alpha amplifies early cyclooxygenase-2 expression, oxidative stress and MAP kinase phosphorylation after cerebral ischemia in mice［J］. J Neuroinflamm, 2010, 7(1):42.

［19］ Wu T, Shi JX, Geng S, et al. The MK2/HuR signaling pathway regulates TNF-α-induced ICAM-1 expression by promoting the stabilization of ICAM-1 mRNA［J］. BMC Pulm Med, 2016, 16(1):84.

［20］ Kotlyarov A, Neininger A, Schubert C, et al. MAPKAP kinase 2 is essential for LPS-induced TNF-alpha biosynthesis［J］. Nat Cell Biol, 1999, 1(2):94-97.

［21］ Wang X, Xu L, Wang H, et al. Mitogen-activated protein kinase-activated protein (MAPKAP) kinase 2 deficiency protects brain from ischemic injury in mice［J］. J Biol Chem, 2002, 277(46): 43968-43972.

［22］ Li Q, Guo YL, Li Zh，et al. The interference of picroside Ⅱon the expressions of Caspase-3 and PARP following cerebral ischemia reperfusion injury in rats［J］. Chinese Pharmacological Bulletin, 2010, 26(3): 342-345. (in Chinese)

李琴, 郭云良, 李震, 等. 胡黄连苷Ⅱ对大鼠脑缺血/再灌注损伤Caspase-3和PARP表达的影响［J］. 中国药理学通报，2010, 26(3): 342-345.

［23］ Zhang HY, Zhai L, Wang TT, et al. Picroside Ⅱ exerts a neuroprotective effect by inhibiting the mitochondria cytochrome C cignal pathway following ischemia reperfusion injury in rats［J］. J Mol Neurosci, 2017, 61(2):267-278.

［24］ Li S, Wang T, Zhai L, et al. Picroside Ⅱ exerts a neuroprotective effect by inhibiting mPTP permeability and EndoG release after cerebral ischemia/reperfusion injury in rats［J］. J Mol Neurosci, 2018, 64(1):144-155.

［25］ Li Sh, Wang TT, Guo YL, et al. Effect of Picroside Ⅱon the expressions of voltage-dependent anion channel 1 after cerebral ischemia reperfusion injury［J］. Chinese Medical Journal, 2017, 98(2):136-142.` (in Chinese)

李珊, 王婷婷, 郭云良, 等. 胡黄连苷Ⅱ对脑缺血再灌注损伤后电压依赖性阴离子通道1表达的影响［J］. 中华医学杂志, 2017, 98(2):136-142.

［26］ Zhao L, Guo YL, Li XD, et al. Effect of Picroside Ⅱon neuron specific enolase after cerebral ischemic injury［J］. Chinese Pharmacological Bulletin, 2014, 30(2):192-199. (in Chinese)

赵丽, 郭云良, 李晓丹, 等. 胡黄连苷Ⅱ对脑缺血损伤后神经元特异性烯醇化酶表达的影响［J］. 中国药理学通报, 2014, 30(2):192-199.

［27］ Wang TT, Zhai L, Zhang HY, et al. Picroside Ⅱ inhibits the MEK-ERK1/2-COX2 signal pathway to prevent cerebral ischemic injury in rats［J］. J Mol Neurosci, 2015, 57(2):335-351.

[28］ Wang TT, Zhai L, Guo YL, et al. Picroside Ⅱ has a neuroprotective effect by inhibiting ERK1/2 activation after cerebral ischemic injury in rats［J］. Clin Exp Pharmacol Physiol, 2015, 42(7):930-939.

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